28 ENZYMES 



Exercise V 



molecules are bound in so-called alpha-linkage, 

 in cellulose in beta-linkage: 



CHoOH 



I 



c— o 



CHoOH 

 C— O 



\ 



Maltose, with 

 two glucose 

 molecules linked 

 as in starch. 



-0- 



V Cellobiose. with 



r^ two glucose 



^ molecules linked 



^ as in cellulose. 



C— C 



C— O 



I 

 CHoOH 



(Write all the missing — H and — OH groups 

 into the above structures.) 



The essential difference between starch and 

 cellulose is therefore one of molecular shape; but 

 that is reason enough for the amylases, which 

 digest starch, to have no effect on cellulose, and 

 for the rare group of enzymes that digest cellu- 

 lose (cellulases) to have no effect on starch. 



When you have time, make the molecular 

 models of maltose and cellobiose, starting in 

 each case with two molecules of glucose. 



Experiments 



Reaction rate vs. enzyme concentration. Stimu- 

 late your flow of saliva by chewing a piece of 

 gum, and collect about 5 ml in a test tube. 

 Working with your partner, make a series of 

 dilutions in tap water as follows: 



Dilution Concentration of saliva 

 (ml saliva: ml tap water) C^) 



1:9 10 



1:19 5 



1:49 2 



1:99 1 



These are conveniently made by taking 1 ml of 

 saliva for each dilution (using a pipet) and 

 making it up to the indicated total by adding 



tap water from a 50-ml graduate. (Do not use 

 the same pipet for the starch solutions.) Meas- 

 ure the activity of these four dilutions of saliva 

 as follows: 



(1) Pipet 1 ml of each concentration of saliva 

 into a test tube and label. 



(2) Into each of a second series of four test 

 tubes pipet 2 ml of 0.59^, starch suspension, 

 made up in 0.259( NaCl. (Salt is added because 

 chloride ions specifically activate salivary amyl- 

 ase.) Add 2 ml of buffer solution, pH 6.8. to 

 each tube (this is the optimal pH for the en- 

 zyme). 



(3) Place the two sets of tubes (eight in all) 

 in the water bath at 37°C. Leave for several 

 minutes until they reach that temperature. (Note: 

 This experiment can also be performed at room 

 temperature but will go more slowly (see next 

 exercise). 



(4) At a recorded time, pour the contents of 

 one tube containing starch mixture into the tube 

 with the highest concentration of saliva. Swirl 

 to mix, and return it quickly to the bath. 



(5) Working with your partner, test for starch 

 by removing a drop of the reaction mixture 

 with a medicine dropper and adding it to a drop 

 of an aqueous solution of lo in KI (each 0.01 

 M) on a test plate. {Note: The lo-KI solution 

 should not be allowed to stand in the test plate 

 depressions for more than a few minutes. Dis- 

 pense it one drop at a time as needed.) These 

 tests should be started at a time as near zero as 

 possible, and continued at 10-second intervals 

 thereafter. The initial color should be blue; 

 continue the tests until they yield no color 

 change at all. Note the colors you see, and 

 record the time required to reach the endpoint, 

 the point at which the mixture has the same 

 color as the iodine test solution. 



(6) Repeat the procedure of steps 4 and 5 

 with the 1:19 dilution. Depending on the rate 

 of reaction, the time intervals between tests can 

 be lengthened. 



